Research Publication Series: July 2018

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Cover Image from “Closed-system Manufacturing of CD19 and Dual-targeted CD20/19 Chimeric Antigen Receptor T cells Using the CliniMACS Prodigy Device at an Academic Medical Center�

I am a Physician-Scientist in the Division of Hematology/ Oncology within the Department of Medicine. I care for adult patients undergoing hematopoietic stem cell transplantation (HSCT). My research lab focuses on the immunobiology of allogeneic stem cell transplantation.

Specifically, we are interested in understanding the pathophysiology of graft versus host disease (GVHD) which is the major complication of allogeneic HSCT. Since GVHD is characterized by the over production inflammatory cytokines, we seek to understand the inflammatory and regulatory networks that modulate this disease. In addition, our lab seeks to translate preclinical findings into the clinic in order to reduce complications from GVHD and improve overall patient survival.

William Drobyski, MD, FACP Professor of Medicine, Pediatrics and Microbiology Mariette C. and Philip W. Orth/Tom Anderson Chair of Oncology Co-Leader, Discovery and Developmental Therapeutics Program, MCW Cancer Center Medical College of Wisconsin

“Tocilizumab, Tacrolimus and Methotrexate for the Prevention of Acute GraftVersus-Host Disease: Low Incidence of Lower Gastrointestinal Tract Disease�

Drobyski WR, Szabo A, Zhu F, et al. Haematologica. 2018;103(4):717-727.

We conducted a phase 2 study in which patients undergoing allogeneic hematopoietic stem cell transplantation received Tocilizumab in addition to standard immune suppression with tacrolimus and methotrexate for graft versus host disease (GVHD) prophylaxis. Thirty-five patients were enrolled between January 2015 and June 2016.

The cumulative incidences of grades II-IV and III-IV acute GVHD were 14% (95% CI 5-30) and 3% (95% CI 0-11) at day 100. A comparison to 130 matched controls who only received tacrolimus and methotrexate demonstrated a lower cumulative incidence of grades II-IV acute GVHD (17% versus 45%, p=0.003) and a significant increase in grades II-IV acute GVHD-free survival at 6 months (69% versus 42%, p=0.001) with Tocilizumab, tacrolimus and methotrexate. We conclude that Tocilizumab has promising activity in preventing acute graft versus host disease and warrants examination in a randomized setting.

Tac/MTX

40 Toc/Tac/MTX 0

0

Cumulative Incidence, %

Months

4

6

100

0

E

80 60 40 Toc/Tac/MTX

20

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0 0

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6 Months

9

12

Tac/MTX

40 20

2

Toc/Tac/MTX

60

20 0

D

80

3

9

6 Months

100 80 60 Tac/MTX

20

Toc/Tac/MTX

0 0

3

6 Months

80 60 Tac/MTX 40 20

9

12

Toc/Tac/MTX

0 0

F

40

100

12

3

6 Months

9

12

9

12

100

Probability, %

60

Probability, %

80

C

100

Cumulative Incidence, %

B

100

Cumulative Incidence, %

A

Cumulative Incidence, %

GVHD and transplant outcomes in patients treated with Tocilizumab versus a matched Center for International Blood and Marrow Transplant-derived control population.

Tac/MTX

80 60

Toc/Tac/MTX

40 20 0 0

3

6 Months

(A). Cumulative incidence of grades II-IV acute GVHD in patients treated with Tocilizumab versus the matched control cohort. (B). Probability of grades II-IV acute GVHDfree survival. Cumulative incidence of (C) chronic GVHD, (D) transplant-related mortality, and (E) relapse. (F) Probability of disease-free survival in patients treated with Tocilizumab versus the matched control cohort.

Jean Pearce, MD, MS Assistant Professor of Pediatrics Division of Emergency Medicine Department of Pediatrics Medical College of Wisconsin

I am a pediatric emergency medicine physician at Children’s Hospital of Wisconsin. I am originally from North Dakota and completed my residency in pediatrics at the University of California Davis Medical Center. I have been at CHW for almost 5 years, completing my fellowship in pediatric emergency medicine here and joining the faculty in 2016. My research interests are two-fold. First, along the lines of this study, I am interested in pediatric procedural sedation including post-sedation home outcomes and modalities for improving patient experience. Second, I am interested in simulation-based medical education and using this for summative assessment of performance.

“Behavioral Changes in Children After Emergency Department Procedural Sedation” Pearce JI, Brousseau DC, Yan K, Hainsworth KR, Hoffman RG, Drendel AL. Academic Emergency Medicine. 2018;25(3):267-274.

This prospective cohort study aimed to determine the proportion of children undergoing ketamine sedation for fracture reduction in the ED who experienced negative post-discharge behaviors and to evaluate predictors of these behaviors. Patient anxiety was assessed presedation using the modified-Yale Preoperative Anxiety Scale. Parents filled out the PostHospitalization Behavior Questionnaire (PHBQ) which assesses negative behavioral changes postprocedure or hospitalization. Sedations were completed and patients were discharged. Parents completed a second PHBQ after discharge, rating their child’s behavior in the 1-2 weeks after sedation. This PHBQ was compared to the baseline to determine behavioral change. 97 patients were enrolled; 82 completed follow-up. 22% (n=18) had negative behavioral changes and 40% (n=33) were highly anxious pre-sedation. The independent predictors for negative behavioral change were high anxiety (OR=9) and non-white race (OR=6.5).

Table 4. Multivariable Analysis: Negative Behavioral Change After ED Procedural Sedation

Xuelin Lou, MD, PhD Associate Professor Department of Cell Biology, Neurobiology & Anatomy Medical College of Wisconsin

As a neuroscientist, I am fascinated with neuro communication and its incredible impact on human brain.

Before joining MCW, I was a faculty member at Yale Medical School and UW-Madison. My group focuses on synaptic transmission, a fundamental process that controls our feeling, memory, decision, and action and deeply implied in multiple brain disorders. We hope to identify new therapeutic targets by uncovering the mechanistic links between synaptic dysfunction and neurological diseases.

We utilize cutting-edge approaches, such as superresolution imaging, electrophysiology and mouse models in our study. Our work has been funded continuously by NIH and published in high-profile journals.

“Sensing Exocytosis and Triggering Endocytosis at Synapses: Synaptic Vesicle Exocytosis-Endocytosis Coupling” Lou X. Frontiers in Cellular Neuroscience. 2018;12:66.

Intact synaptic nanostructure is critical for information processing in neural circuits. During synaptic transmission, rapid vesicle exocytosis increases the size of never terminals and endocytosis counteracts it. Synaptic vesicle exocytosis and endocytosis are tightly connected in time and space; however, the mechanisms that timely connect these two fundamental events are poorly understood. Addressing this question is essential to understanding how neurons precisely control their communication and plasticity at molecular and cellular levels. Based on our work, as well as recent findings from others, we proposed several ideas by which synapses can "sense" the occurrence of exocytosis and timely initiate compensatory endocytosis. They include Ca2+ sensing, SV proteins sensing, and local membrane stress sensing. To test these models, much work is required by incorporating the latest innovative research technology in this field.

Figure 1. The transient expansion of the presynaptic surface area.

(A) Ultrastructure of the presynaptic nerve terminals in a mouse cerebellum (chemical fixation). Note two AZs with high electronic density (between the arrowheads). (B) The surface area of a single SV after it fuses and merges with the PM at an AZ. The scheme size is shown in scale, the AZ area is 0.4 μm2 (Holderith et al., 2012) and SV diameter is 45 nm (Lou et al., 2008). (C) Transient changes of the surface area are recorded at the calyx of Held during a 20 ms depolarization pulse (arrow) (Lou et al., 2008). Note the rapid increase and subsequent recovery of Cm, suggesting SV fusion and compensatory endocytosis.

Fenlu Zhu, PhD Assistant Professor of Medicine Division of Hematology/Oncology Assistant Director, BMT Laboratories Medical College of Wisconsin

I am the Assistant Director of Blood and Marrow Transplant Laboratories and an Assistant Professor in the Division of Hematology/Oncology, Department of Medicine at MCW. My research interests and expertise are preclinical process development and validations; large-scale cGMP manufacturing of cellular products including patient cell purification, expansion, transduction and formulation; product characterization and release testing such as phenotype, potency, purity and stability; technology transfer from research bench to clinical production; immune monitoring after cellular therapy and correlative studies. I am also knowledgeable in FACT, CAP and FDA regulations.

“Closed-system Manufacturing of CD19 and Dual-targeted CD20/19 Chimeric Antigen Receptor T cells Using the CliniMACS Prodigy Device at an Academic Medical Center” Zhu F, Shah N, Xu H, et al. Cytotherapy. 2018;20(3):394-406.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable efficacies in treating B-cell hematologic malignancies. Two CAR-T live drugs have been approved by FDA in 2017. But the current centralized manufacturing model has posed logistic challenges and the manufacturing process is complex requiring skilled personal and costly clean-room facilities. To help making this effective therapy available to more patients, we employed Miltenyi CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction. With this system, we are able to manufacture sufficient CAR-T cells with potent antitumor activity for clinical use.

Figure 6. Effect of CD56 depletion on the lysis of parental K562 targets.

(A) CAR-T-cell line immediately after harvest. (B) Same line after depletion of CD56+ cells. Fewer than 0.1% CD56+ cells remained in the culture post-depletion. Targets are represented by the following symbols. Raji B cell line, square; K562CD19, triangle; K562CD20, inverted triangle; K562-parental, circle; autologous T cells, diamond.

Figure 8. Effects of thawing and expansion on CAR-T cells.

(A) Effector function at harvest. (B) Cells after 7 days of culture. (C) Percentage of the total cells that were transduced CD3+, CD4+ or CD8+ T cells.

Targets are represented by the following symbols. Raji B cell line, square; K562CD19, triangle; K562-CD20, inverted triangle; K562parental, circle; autologous T cells, diamond. The intracellular cytokine data for the indicated stimulus is expressed as the percentage of IFN-γ produced by gated CD8+ cells (top bar) and CD4gated T cells (lower bar).

Rebekah J. Walker, PhD Assistant Professor of Medicine Division of General Internal Medicine Center for Advancing Population Science Medical College of Wisconsin

I am an Assistant Professor and Lead for the Health Systems Research Unit in the Center for Advancing Population Science at MCW. My research focuses on reducing and eliminating health disparities by addressing social determinants of health. I completed my PhD at the Medical College of South Carolina in health services, focusing my dissertation on the impact of social determinants of health on diabetes outcomes. I was trained in conducting analyses of outcome and cost-effectiveness studies and gained experience with large retrospective dataset analysis, and advanced statistical techniques. My research focuses on the psychosocial, behavioral, and community factors influencing low income populations, with particular emphasis on factors such as stress, food insecurity, and segregation in populations with chronic disease.

“Racial Differences in Spatial Patterns for Poor Glycemic Control in the Southeastern United States�

Walker RJ, Neelon B, Davis M, Egede LE. Annals of Epidemiology. 2018;28(3):153-159.

The purpose of this study was to identify hotspots of racial disparities in prevalence and control of diabetes using novel geospatial analytical methods. Using 64,022 non-Hispanic Black and nonHispanic White Veterans with diabetes in the southeastern US, glycemic control was categorized as uncontrolled if HbA1c>=8%. Analyses included logistic regression with spatial random effects, and Moran’s I hotspot analysis on county level aggregated data. Hotspots for poor glycemic control differed by race, and spatial patterns provided additional explanatory power on the distribution of racial disparities, but the role of race persisted. Given the VA provides more equal access than the overall US healthcare system, results suggest the built environment explains some of the disparities in diabetes and suggests unmeasured factors related to race need to be examined in future studies.

Figure 1. Percent of population with uncontrolled HbA1c by county

Figure 2. Hotspots for uncontrolled HbA1c in full sample, nonHispanic whites, and non-Hispanic blacks

“Modeling Protein Aggregation and the Heat Shock Response in ALS iPSCDerived Motor Neurons” Seminary ER, Sison SL, Ebert AD. Frontiers in Neuroscience. 2018;12:86.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous neurodegenerative disease caused by the loss of the upper and lower motor neurons. While many studies have aimed at understanding the pathologies associated with the most prevalent mutations, few have attempted to study multiple mutations at once. Here, we show that motor neurons generated from patient iPSCs carrying mutations in three different common genes all exhibited protein aggregation, although activation of the protein quality control response pathways designed to limit these inclusions was variable across the genotypes. These data support the use of protein quality control agonists as a potential therapy for ALS.

Emily Seminary Graduate Student Department of Cell Biology, Neurobiology & Anatomy: Ebert Lab

Farshad Mostafaei, PhD Postdoctoral Fellow Department of Radiation Oncology

“Real-Time Motion Tracking Using Ultrasound and Intrafractional KV Cone Beam Projection Images” Mostafaei F, Tai A, Haase W, et al. Medical Physics. 2017;44(6):3269-3270.

To investigate the feasibility of real-time tracking lung tumor motion using continuous 2D ultrasound (US) and periodic 2D x-ray images created from cone beam projection images (CBPI). Time-sequenced b-mode US and cone CBPI data were extracted from the Clarity® and XVI platforms on an Elekta linac. The data was synchronized through a video capture card (VCE-PRO, IMPERX Inc.) which was triggered by the XVI system. In this way, a system was configured to allow real-time acquisition of the diaphragm position synchronized with periodic acquisition of the lung tumor position. Feasibility of the system was demonstrated by acquiring synchronized data from ultrasound images of diaphragm and CBPI of lung tumor on three lung cancer patients consented to participate in the study. The data were acquired in two fractions for each patient.

“Preoperative Pain in Patient with an Inguinal Hernia Predicts Long-term Quality of Life” Mier N, Helm M, Kastenmeier AS, Gould JC, Goldblatt MI. Surgery. 2018;163(3):578-581.

We hypothesized that patients presenting with less pre-operative pain would experience a greater improvement in long-term quality of life after an inguinal hernia repair. Patients underwent either laparoscopic or open inguinal hernia repair and physical/mental component scores (PCS and MCS) were calculated from the SF-12 surveys administered preoperatively, at six months, and twelve months. Patients also completed an analog surgical pain scale. Patients who reported no or a low amount of preoperative pain experienced improved PCS quality of life compared with patients who reported moderate to severe preoperative pain (P = .048). This relationship was not noted with MCS.

Neil Mier, BS 3rd Year Medical Student Department of Surgery

Karlo Kovacic, MD, MSc Pediatric Gastroenterology Fellow PGY-4 Division of Gastroenterology, Hepatology & Nutrition

“Healthcare Utilization and Comorbidities Associated with Anorectal Malformations in the United States” Kovacic K, Matta SR, Kovacic K, Calkins C, Yan K, Sood MR. The Journal of Pediatrics.

2018;194:142-146.

The objective of our study was to determine nationwide prevalence and healthcare utilization in children with anorectal malformations and associated anomalies over a 6-year period. We identified close to 2400 children <2 years of age with anorectal malformations.

Other congenital anomalies were reported in 80% of anorectal malformations and were closely associated with increased length of stay and costs. The average annual healthcare expenditure for surgical correction of anorectal malformations and associated anomalies for the years studied was US $45.5 million. This study showed us that high complexity and need for lifelong multidisciplinary management is associated with substantial healthcare expenditure.